Normal adherent cells require soluble factors and adhesion to the extracellular matrix (ECM) for cell cycle progression. Integrins are adhesion receptors that bind to the ECM and can participate in signaling for cell cycle control. The cyclin-dependent kinase inhibitor p21 (CIP1/WAF1) blocks cell cycle progression. Our laboratory has demonstrated that p21 is regulated in two discrete phases during G1 phase of the cell cycle. Early G1 induction of p21 gene expression is mitogen-dependent but independent of integrin signaling. However, during mid-late G1, p21 gene expression is downregulated in an integrin-dependent manner. This effect can contribute to the activation of cyclin E-cdk2 complexes and progression into S phase. The overall goal of this proposal is to examine the integrin-mediated mechanisms involved in the repression of the p21 promoter. Aim 1 uses a constitutively active MEK vector (which induces p21 expression) and promoter luciferase assays to determine whether integrins require coordinate signaling with receptor tyrosine kinases for the downregulation of the p21 promoter. Aim 2 uses expression of dominant negative and constitutively active Rho constructs, integrin clustering experiments, and promoter luciferase assays to determine the role of Rho in the integrin-dependent downregulation of the p21 promoter. Based on results in Aim 1 and 2, Aim 3 will use dominant negative or constitutively active mutants of specific Rho effectors or FAK to elucidate the integrin-dependent mechanisms involved in the downregulation of p21. The long-term goal is to elucidate a mechanism that will help us understand how regulation of the cell cycle becomes uncoupled from cell adhesion during cellular transformation.